The ETS family of transcription factors regulates the expression of thousands of genes, both housekeeping and tissue-specific. The ETS DNA binding domain, or simply, the ETS domain, is conserved throughout the family;however, outside of the ETS domain the family is diverse and specialized allowing the ETS factors to target and control transcription in unique ways. These include autoinhibitory mechanisms that control DNA binding affinity and interactions with co-factors that control gene expression. A recent discovery in prostate cancer revealed that a sub-set of ETS proteins (ERG, ETV1, ETV4, or ETV5) undergo chromosomal translocations in 50% of these cancers and the misregulation of these ETS factors is important for transformation and cancer progression. The goal of this proposal is to reveal the unique features of the ETS factors involved in prostate cancer to not only further our understanding of the ETS family, but to provide biochemical details important for designing prostate cancer therapeutics. Successful completion of the work will include a thorough understanding of allosteric regulatory mechanisms of ETS factors involved in prostate cancer and discovery and characterization of nuclear co-factor interactions with these oncogenic proteins. It is unknown if autoinhibition controls ERG, ETV1, ETV4 or ETV5, however, evidence throughout the ETS family suggests modulating DNA binding through inhibitory domains is a feasible mechanism for regulating ETS DNA binding. This can easily be measured by assessing DNA binding affinities using EMSAs. Truncation mutants will be generated in order to pinpoint regions involved in the inhibition and to biochemically assess the nature of the autoinhibition. Finally, inhibitory domains will be characterized structurally using well established NMR techniques. In conjunction, an unbiased approach will be used for capturing interacting partners both within cells and in vitro from cellular nuclear extracts. Mass spectrometry technologies will allow positive identifications of ETS interacting partners, which will be further, validated using biochemistry. Limited proteolysis will identify specific domains that mediate the interaction and simple reporter assays will be used to probe for the function of the interaction within cells. To realize the full potential of understanding new co-factors, the structures of these interfaces will be determined by NMR techniques. PUBLIC HEALTH RELEVANCE: Human chromosomal rearrangements that affect ETS factors are involved in both Ewing's sarcoma and prostate cancer. The completion of the proposed experiments will provide a detailed picture of the regulatory mechanisms controlling the ETS factors involved in prostate cancer, ERG, ETV1, ETV4 and ETV5, including both inhibitory properties and co-factor interactions. Both of these interfaces are important to understand from an ETS family perspective, but can also be exploited for rational drug design in the treatment of prostate cancer.